Compositions and kits for detecting a sequence mutation in cinnamyl alcohol dehydrogenase gene associated with altered lignification in loblolly pine

ABSTRACT

Loblolly pine ( Pinus taeda  L.) is the most important commercial tree species in the USA harvested for pulp and solid wood products. Increasing the efficiency of chemical pulping may be achieved through the manipulation of genes involved in the lignin biosynthetic pathway. A null allele of cinnamyl alcohol dehydrogenase (CAD) has been discovered in the loblolly pine clone 7-56 which displays altered lignin composition. During identification of single nucleotide polymorphisms (SNPs) in the cad gene, a two-base pair adenosine insertion located in exon five and unique to clone 7-56 was discovered. The sequence mutation causes a frame-shift predicted to result in premature termination of the protein. For routine detection of the mutation, a diagnostic assay was developed utilising Template-directed Dye-terminator Incorporation and Fluorescence Polarization detection (FP-TDI).

RELATED APPLICATION

The present application is a divisional application of U.S. patentapplication Ser. No. 10/359,451 filed Feb. 5, 2003 now U.S. Pat. No.6,921,643.

GOVERNMENT INTEREST

This invention was made with United States support under Grant No.9975806 awarded, by the National Science Foundation. The United Stateshas certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of pine tree breeding and selection. Inparticular, this invention relates to methods and compositions foridentifying pine trees that harbor the null cinnamyl alcoholdehydrogenase (CAD) allele (cad-n1).

BACKGROUND OF THE INVENTION

Global consumption of wood products is projected to increase 25% overcurrent levels by 2015 (McLaren 1999). Full citations for the referencescited herein are provided before the claims. Forest plantations areincreasingly important to meet these global demands because their fastergrowth rates result in much more harvestable volume per unit area thannatural forests (Hagler 1996, Sedjo 1999). Thus, reliance on plantationsreduces the need to harvest natural forests, allowing them to be usedfor other societal purposes. In fact, as little as 5 to 10% of the totalarea of world's forests would be required to meet global demands forwood products if this area were devoted to fast-growing plantations(Hagler 1996, Sedjo and Botkin 1997). Further, the faster growth ratesmean high rates of carbon sequestration that may mitigate the effects ofglobal warming. These facts, coupled with the declining area availablefor commercial forest harvests due to deforestation and governmentrestrictions, have led to a global effort to increase plantation growthrates per unit area above current values through both classical and newtechnologies (Fox 2000).

Viewed as an agricultural crop, timber is the single highest-valued cropin the USA and loblolly pine (Pinus taeda L) is the most importantcommercial tree species in the USA. Each year more than 900 millionseedlings are used to establish loblolly pine plantations on more thanhalf a million hectares (Pye et al. 1997). The total acreage of theloblolly pine plantation estate is estimated at more than 12 millionhectacres (Byram et al. 1999). Loblolly pine is also important for itsecological and biological importance in native forests. Its native rangespans 14 states from southern New Jersey south to central Florida andwest to Texas. In these natural forests it is the dominant tree specieson 11.7 million ha (Baker and Langdon 1990). Thus, loblolly pine isnearly equal in its distribution between native and planted foreststotaling 23.7 million hectares. By comparison, the total expanse ofplantations of hybrid poplar in the Pacific Northwest is approximately25,000 ha (Nuss 1999), which is only 0.2% of the area planted inloblolly pine.

Due to its overwhelming commercial importance, tree breeding programsfor loblolly pine began in the 1950's, and virtually all forest productscompanies and state agencies are involved in genetic improvementprograms (more than 30 organizations) (Byram et al. 1999, Li et al.1999). These programs have used classical methods of selection, genetictesting and breeding to make demonstrable genetic progress.Unfortunately the progress is hindered, compared to that in agriculturalcrops, by the large size and long-lived nature of pines (eight years infield tests to make selections followed by another five or more years tocomplete breeding). For these reasons, most loblolly pine programs areonly in their second or third cycle of breeding after nearly 50 years,when in some crops more than one cycle is completed in a single year.

Loblolly pine (Pinus taeda L.) is the most intensively grown treespecies in the USA for pulp and solid wood products with plantationsexceeding 12 million hectares. The extraction of lignin from wood duringthe production of pulp and paper requires the use of costly chemicalsthat are toxic to the environment. Significant progress towardsincreasing pulping efficiency has been achieved in poplar through thegenetic manipulation of genes involved in lignin biosynthesis (Baucheret al., 1996, Hu et al., 1999; Pilate et al., 2002). One of the keyenzymes successfully targeted, cinnamyl alcohol dehydrogenase (CAD),catalyzes the final step in the synthesis of monolignols by convertingcinnamaldehydes to cinnamyl alcohols. Field-grown transgenic poplar withreduced-CAD allowed easier delignification, using smaller amounts ofchemicals and yielded more high quality pulp without an adverse effecton growth (Pilate et al., 2002).

A null CAD allele (cad-n1) has been discovered in the loblolly pineclone 7-56 which is heterozygous for the null allele (MacKay et al.,1997). Homozygous seedlings (cad-n1/cad-n1) obtained by selfing, containbetween 0-1% of wild type CAD activity (MacKay et al., 1997) and displaya brown-red wood phenotype. The expression level of cad transcript inshoot, megagametophyte and xylem tissues was 20 times less in cad-n1homozygous plants compared to wild type (MacKay et al., 1997).

Deficiency of CAD in cad-n1 homozygotes only slightly reduces lignincontent but drastically alters lignin composition (MacKay et al., 1997;Ralph et al., 1997; Lapierre et al., 2000; MacKay et al., 2001). Themajor lignin composition change was attributed to the incorporation ofdihydroconiferyl alcohol (DHCA), a minor component of most lignins, butelevated to levels 10-fold higher in cad-n1 homozygous trees.Coniferaldehyde, the substrate of CAD, and vanillin are also present inincreased levels while the coniferyl alcohol component of normal lignindecreased.

The mutation has a variable effect on pulping efficiency, depending onthe age of the trees and whether the mutation is present in a homozygousor heterozygous state. In totally CAD-deficient trees (cad-n1/cad-n1),delignification was significantly easier but the pulp yields wererelatively low (˜33%) compared to normal trees (48%) (Dimmel et al.,2001). In 4-6 year old partially CAD-deficient trees (heterozygous)delignification increased in efficiency by ˜20% and yields were similarto wild type (Dimmel et al., 2002). In contrast to these younger trees,a small sample of 14 year old partially CAD-deficient trees displayed nomajor differences in ease of delignification and pulp yield (Dimmel etal., 2002).

In addition to lignin composition changes, the cad-n1 allele appears tobe associated with increased stem-growth traits in heterozygous trees(Wu et al., 1999). This growth promotion correlates to an increase indebarked volume of 4-year old trees (14%) (Wu et al., 1999) that is alsoobserved in 14-year old trees (Dimmel et al., 2002). A likelyexplanation could be that trees harboring the cad-n1 allele may investfewer resources into the production of monolignols, allowingreallocation of resources towards growth. Promotion of growth was alsoobserved in transgenic poplar with the lignin biosynthetic enzyme4-coumarate:coenzyme A ligase (4CL) down-regulated (Hu, et al., 1999).

For the above reasons, it is desirable to be able to select pine treesthat harbor the null CAD allele (cad-n1). Traditionally, the mutationhas been diagnosed using CAD isozyme analysis on haploidmegagametophytes obtained from seed or by using genetic markers closelylinked to the mutation (MacKay et al., 1997). These prior art methodsare slow and tedious. It takes numerous years for pine tree seedlings toproduce suitable seed for CAD isozyme marker analysis. In addition,linked genetic marker analysis is slow and often yields inaccurateresults. There is thus a tremendous need to develop methods that allowrapid and accurate identification of pine trees that harbor the null CADallele (cad-n1).

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention relates to theidentification of a sequence mutation responsible for the loss offunction associated with the cad-n1 allele. This mutation was identifiedduring single nucleotide polymorphism (SNP) discovery within the cadgene of loblolly pine. Identification of this mutation allows breedersto accurately determine the presence, absence and/or copy number of thecad-n1 allele in their germplasm before it reaches sexual maturity.

The present invention is directed to a method of identifying a loblollypine tree harboring a null CAD allele (cad-n1) wherein the pine treecontains a cad gene and the cad gene has a fifth exon. A pine tree issaid to “harbor” or contain the null CAD allele if it is homozygous forthe null CAD allele (cad-n1/cad-n1) or is heterozygous for the null CADallele (cad-n1/cad). Pine trees that are homozygous for the wild typeCAD allele (cad/cad) do not harbor the null CAD allele. This sequencediffers from the wild type sequence of the fifth exon of the cad genedepicted in SEQ ID NO:1. It is expected that there will be some geneticvariation in the wild type cad gene sequence resulting in slightdifferences in the wild type sequence compared to SEQ ID NO:1.

In one format, the method includes identifying a pine tree containing atwo base pair adenosine insertion in the fifth exon of the cad genewherein the DNA sequence of the two base pair adenosine insertionincludes the nucleotide sequence depicted in SEQ ID NO:3 or thecomplement thereof.

The present invention is further directed to a method of selecting aloblolly pine tree harboring a null CAD allele (cad-n1) wherein the pinetree contains a cad gene and the cad gene has a fifth exon. The methodincludes a) providing a sample including DNA from the pine tree whereinthe DNA includes the cad gene; b) determining whether the fifth exoncontains a two base pair adenosine insertion wherein the nucleotidesequence of the fifth exon containing the two base pair adenosineinsertion includes the nucleotide sequence depicted in SEQ ID NO:3 orthe complement thereof wherein the identification of the two base pairadenosine insertion is indicative of a pine tree harboring a null CADallele (cad-n1) and c) identifying a sample containing the two base pairadenosine insertion to thereby select a loblolly pine tree harboring anull CAD allele (cad-n1).

The present invention is further directed to a method of identifying aloblolly pine tree harboring a null CAD allele (cad-n1) wherein themethod includes a) providing a sample including DNA from the pine treewherein the DNA contains a cad gene and the cad gene has a fifth exon;b) performing template-directed dye-terminator incorporation andfluorescence polarization detection (FP-TDI) on the DNA to determinewhether the fifth exon in the sample contains a two base pair adenosineinsertion wherein the nucleotide sequence of the fifth exon containingthe two base pair adenosine insertion includes the nucleotide sequencedepicted in SEQ ID NO:3 wherein the two base pair adenosine insertion isindicative of a pine tree harboring a null CAD allele (cad-n1) and c)selecting a sample containing the two base pair adenosine insertion inthe cad gene to thereby identify a loblolly pine tree harboring a nullCAD allele (cad-n1).

The present invention is further directed to a method of identifying aloblolly pine tree harboring a null CAD allele (cad-n1) by firstproviding a sample including DNA from the pine tree wherein the DNAcontains a cad gene and the cad gene has a fifth exon wherein the DNA inthe sample is amplified by PCR using PCR primers wherein the sequencesof the primers is SEQ ID NO:11 and SEQ ID NO:12. Next, template-directeddye-terminator incorporation and fluorescence polarization detection(FP-TDI) is performed on the DNA using oligonucleotides havingnucleotide sequences SEQ ID NO:13 and SEQ ID NO:14 to determine whetherthe fifth exon of the cad gene in the sample contains a two base pairadenosine insertion wherein the nucleotide sequence of the fifth exoncontaining the two base pair adenosine insertion includes the nucleotidesequence depicted in SEQ ID NO:3 wherein the two base pair adenosineinsertion is indicative of a pine tree harboring the null CAD allele(cad-n1). Finally, samples are selected containing the two base pairadenosine insertion in the cad gene to thereby identify a loblolly pinetree harboring a null CAD allele (cad-n1).

The present invention is further directed to a method of identifying aloblolly pine tree homozygous for the null CAD allele (cad-n1/cad-n1)wherein the pine tree contains a cad gene and the cad gene has a fifthexon, by identifying a pine tree, wherein the pine tree contains DNAwith a two base pair adenosine insertion in the fifth exon of the cadgene wherein the DNA sequence of the two base pair adenosine insertionincludes the nucleotide sequence depicted in SEQ ID NO:3 or thecomplement thereof. In this format, the selected pine tree does notcontain DNA with wild type sequence for the fifth exon of the cad genewherein the wild type sequence is depicted in SEQ ID NO:1.

The present invention is further directed to a method of identifying aloblolly pine tree heterozygous for the null CAD allele (cad/cad-n1)wherein the pine tree contains a cad gene and the cad gene has a fifthexon, by identifying a pine tree, wherein the pine tree contains DNAwith a two base pair adenosine insertion in the fifth exon of the cadgene wherein the DNA sequence of the two base pair adenosine insertionincludes the nucleotide sequence depicted in SEQ ID NO:3 or thecomplement thereof. In this format, the pine tree also contains wildtype sequence for the fifth exon of the cad gene wherein the wild typesequence is depicted in SEQ ID NO:1 or the complement thereof.

The present invention is further directed to a method of identifying aloblolly pine tree homozygous for the wild type CAD allele (cad/cad)wherein the pine tree contains a cad gene and the cad gene has a fifthexon by identifying a pine tree, wherein the pine tree lacks DNA with atwo base pair adenosine insertion in the fifth exon of the cad genewherein the DNA sequence of the two base pair adenosine insertionincludes the nucleotide sequence depicted in SEQ ID NO:3 or thecomplement thereof to thereby identify a pine tree homozygous for thewild type CAD allele (cad/cad).

In the methods of the invention, the identifying step may be performedon a sample isolated from a pine tree, a pine tree seedling, a pine treetissue culture, a pine tree cell culture or a pine tree megagametophte.The sample may also be from pine bark, pine needle, pine tissue or pineseed.

In the methods of the invention, the two base pair adenosine insertionmay be identified by any genotyping assay that relies on the detectionof the presence or absence of the double adenosine insertion mutation.Such methods include DNA sequencing, PCR assays and single base pairextension assays.

The single base pair extension assay may be template-directeddye-terminator incorporation and fluorescence polarization detection(FP-TDI).

In one format of the invention, the FP-TDI assay may include the use ofoligonucleotides wherein the sequences of the oligonucleotides are SEQID NO: 13 or SEQ ID NO: 14. The FP-TDI assay may also include the use ofPCR to amplify DNA prior to the FP-TDI assay. In the PCR assay,oligonucleotides such as those depicted in SEQ ID NO:11 and SEQ ID NO:12may be utilized.

The present invention is further directed to an isolated oligonucleotidehaving a nucleotide sequence selected from SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14.

In another format, the present invention is directed to a kit for thedetection of the null CAD allele (cad-n1) in loblolly pine. The kit mayinclude an oligonucleotide such as SEQ ID NO:13 or SEQ ID NO:14.

The kit may further include materials to perform PCR reactions. Suchmaterials to perform PCR reactions may include PCR primers such as thosedepicted in SEQ ID NO:11 and SEQ ID NO:12. The kit may further includeone or more buffers. The kit may also include directions for using thekit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the position of the cad-n1sequence mutation within the cadgene and the effect of the frame-shift on amino acid sequence. A portionof the wild type cad DNA sequence is depicted as SEQ ID NO:1 with thecorresponding amino acid sequence depicted as SEQ ID NO:2. A portion ofthe cad-n1 DNA sequence is depicted as SEQ ID NO:3 with thecorresponding amino acid sequence depicted as SEQ ID NO:4.

FIG. 2 shows a single base extension assay design for both the forwardand reverse reactions. Forward (1528F) and reverse (1528R) assay primerpositions and the corresponding fluorescent dideoxynucleotide terminatorincorporated for the wild type and cad-n1 allele are also depicted. Thesequences depicted in the figure are SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8.

FIG. 3 shows the detection of the cad-n1 sequence mutation in 96 samplesanalyzed by the forward and reverse Template-directed Dye-terminatorIncorporation and Fluorescence Polarization detection (FP-TDI) assay.Plants are grouped as control (heterozygous), control (homozygous wildtype), control (homzygous null), negative controls and unknown plants.

DETAILED DESCRIPTION OF THE INVENTION

Loblolly pine clone 7-56 is heterozygous for the null cad allele(cad/cad-n1) (MacKay et al., 1997). Selfing of these heterozygous 7-56clones produce 25% homozygous mutant seedlings: (cad-n1/cad-n1), 50%heterozygous seedlings (cad/cad-n1) and 25% homozygous wild typeseedlings: (cad/cad). The homozygous cad-n1 seedlings contain between0-1% of wild type CAD activity. Field-grown transgenic poplar withreduced-CAD allows for easier delignification, using smaller amounts ofchemicals and yields more high quality pulp without an adverse effect ongrowth. As such, loblolly pine tree breeders have a stong interest inbeing able to rapidly identify such cad-n1 homozygous plants. It wouldbe particularly useful if a mutation in the cad gene could be identifiedthat was associated with the reduced CAD activity in homozygous plants.Identification of such a mutation would enable the use of various rapidmolecular genetic assays for the identification of (cad-n1/cad-n1),(cad/cad-n1) and (cad/cad) trees and seedlings. The present invention isdirected to methods and compositions useful for indentifying anddistinguishing (cad-n1/cad-n1), (cad/cad-n1) and (cad/cad) trees andseedlings.

As discussed in the Example, SNP discovery within the cad gene wasperformed on haploid megagametophyte DNA from clone 7-56 and 31 otherunrelated individuals. A two-base pair adenosine insertion wasidentified unique to clone 7-56, known to be deficient in CAD activity.The insertion was located in the second codon of exon five and creates aframe-shift that generates a premature stop codon (FIG. 1). Seventeenhaploid megagametophytes from the heterozygous 7-56 clone were assayedby isozyme gel electrophoresis and DNA sequence analysis to confirm thesequence mutation discovered was associated with CAD-deficiency. Inevery case, the two-base pair adenosine insertion corresponded with theabsence of CAD activity and therefore provides a means for rapidlyidentifying and distinguishing (cad-n1/cad-n1), (cad/cad-n1) and(cad/cad) trees and seedlings.

Plants homozygous for the null cad allele (cad-n1/cad-n1) will containDNA having the two base adenosine insertion in the fifth exon of the cadgene (at positions 4 and 5 of SEQ ID NO:3) but will not contain wildtype DNA for the fifth exon of the cad gene as depicted in SEQ ID NO:1.As such, these plants harbor or contain the null CAD allele but do notharbor or contain the wild type CAD allele.

Plants homozygous for the wild type cad allele (cad/cad) will notcontain DNA having the two base adenosine insertion in the fifth exon ofthe cad gene (at positions 4 and 5 of SEQ ID NO:3) but will instead onlycontain wild type DNA for the fifth exon of the cad gene as depicted inSEQ ID NO:1. Such plants do not harbor or contain the null CAD allelebut do harbor the the wild type CAD allele.

Plants heterozygous for the null cad allele (cad-n1/cad) will containDNA having the two base adenosine insertion in the fifth exon of the cadgene (at positions 4 and 5 of SEQ ID NO:3) and will also contain wildtype DNA for the fifth exon of the cad gene as depicted in SEQ ID NO:1.As such, these plants harbor both the null CAD allele and the wild typeCAD allele.

The two-base pair adenosine insertion (at positions 4 and 5 of SEQ IDNO:3) or lack thereof (the wild type sequence, SEQ ID NO:1) can berapidly identified by numerous methods well known to those of skill inthe art. Such methods include any genotyping assay that relies on thedetection of the presence or absence of the double adenosine insertionmutation. Such methods include but are not limited to PCR amplificationreactions, single base extension assays, primer extension assays, DNAsequencing assays and assays utilizing molecular probes [i.e. Taqman &Fluorescence Resonance Energy Transfer, (FRET)] assays and othertechniques.

Primer extension is a simple, robust technique for analyzing singlenucleotide polymorphisms (SNPs) such as the two base pair adenosineinsertion in SEQ ID NO:3 or the complement thereof. This process isillustrated in FIG. 2 and in the Example. A primer with its 3′ enddirectly flanking the SNP is annealed to the amplified target andinduced to extend by a single ddNTP complementary to the polymorphicbase. Based on the molecular weight difference between ddNTPs, extensionproducts vary in weight depending on the incorporated nucleotide. Suchextension products can be correlated and identified with a particularsequence and then utlized to detect the particular sequence.

DNA sequencing is a technique utilized to determine the sequence ofnucleotides in a particular DNA molecule such as the presence or absenceof the two base pair adenosine insertion in SEQ ID NO:2. Typicalsequencing reactions include appropriate sequencing buffers,nucleotides, dideoxy nucleotides, DNA polymerase and one or moreoligonucleotide primers. Clones containing the 5th exon of the cad genecan be sequenced with sequencing primers that flank the cloned insert,e.g. T7 polymerarse primers. Alternatively, PCR products containing the5th exon of the cad gene, prepared, for example, as described below, canbe sequenced directly.

The polymerase chain reaction (PCR) is a technique utilized to amplifyDNA and can be utlized to detect differences in sequences such as thetwo base pair adenosine insertion in SEQ ID NO:3 of the complementthereof. Typical PCR reactions include appropriate PCR buffers,nucleotides, DNA polymerase and one or more oligonucleotide primers. Anyprimer amplifying exon 5 of the cad gene can be utilized. Such primerscan be designed by procedures well known in the art, for example thoseprocedures described on the UK Human Genome Mapping Project ResourceCentre web site. The primers may be located within 3000 base pairs ofexon 5 in pine DNA. Generally, primers should be at least 18 nucleotidesin length to minimize the chances of encountering problems with asecondary hybridization site on the vector or insert. Primers with longruns of a single base should generally be avoided. It is generallyimportant to avoid 4 or more G's or C's in a row. For cycle sequencing,primers with melting temperatures in the range 52-58 degrees C., asdetermined by the (A+T)2+(C+G)4 method, generally produce better resultsthan primers with lower melting temperatures. Primers with meltingtemperatures above 65 degrees C. should also be avoided because ofpotential for secondary annealing. If the template is a high “G-C”templates, then a primer with a Tm in the 60-70 degrees C. range may bedesirable. It is then advisable to do the sequencing reaction withannealing and extension at 60 C. Primers generally have a G/C contentbetween 40 and 60 percent. For primers with a G/C content of less than50%, it may be necessary to extend the primer sequence beyond 18 basesto keep the melting temperature above the recommended lower limit of 50degrees C. Primers should be “stickier” on their 5′ ends than on their3′ ends. A “sticky” 3′ end as indicated by a high G/C content couldpotentially anneal at multiple sites on the template DNA. A “G” or “C”is desirable at the 3′ end but the first part of this rule should apply.Primers should not contain complementary (palindromes) withinthemselves; that is, they should not form hairpins. If this stateexists, a primer will fold back on itself and result in an unproductivepriming event that decreases the overall signal obtained. Hairpins thatform below 50 degrees C. generally are not such a problem. Primersshould generally not contain sequences of nucleotides that would allowone primer molecule to anneal to itself or to the other primer used in aPCR reactions (primer dimer formation). If possible, it is generallyuseful to run a computer search against the vector and insert DNAsequences to verify that the primer and especially the 8-10 bases of its3′ end are unique.

Specific PCR primers, such as those depicted as SEQ ID NO:11 and SEQ IDNO:12, may be utilized in the reaction. Reaction products can besequenced as described above or separated by gel electrophoresis, e.g.size gel electrophoresis, to identify those pine trees harboring or notharboring the CAD null allele.

Various modifications of general DNA sequencing, PCR and primerextension techniques are possible as detailed in Short Protocols inMolecular Biology, 4th Edition ed. F. M. Ausubel, R. Brent, D. D. Moore,K. Struhle, Massachusetts General Hospital and Harvard Medical School(2001) Molecular Cloning, Molecular Cloning, Sambrook et al. (2000) bothof which are hereby incorporated by reference.

While specific oligonucleotide primer sequences are described herein, itis understood that substantially identical oligonucleotide primersequences to those described herein will also work to permit detectionof the two base pair adenosine insertion in SEQ ID NO:3 or thecomplement thereof that is absent from SEQ ID NO:1. The term“substantially identical” oligonucleotide primer sequences means that aoligonucleotide primer comprises a sequence that has preferably at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably atleast 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%,97%, 98%, or 99% sequence identity, compared to a referenceoligonucleotide sequence using standard alignment programs usingstandard parameters.

Pine Tree Plant Material

The two base pair mutation identifying the mutant cad gene can bedetected in pine DNA or possibly RNA from pine tissue, pine cells, orpine cellular extracts. Such pine tissue, pine cells, or pine cellularextracts can be isolated from pine trees, pine tree seedlings, pine treecell culture material, pine tree tissue culture material, pine treeseeds, pine tree needles, bark, tissue and pine tree megagametophytes.Pine seeds, tissue and wood samples can be isolated as described inMacKay, et al. Mol. Gen. Genet. 247, 537-545 (1995) which is herebyincorporated by reference in its entirety. DNA can be extracted frompine needles and megagametophytes as described in Doyle, et al. Focus12, 13-15 (1987) which is hereby incorporated by reference in itsentirety.

Kits

The present invention is also directed to a kit for the rapid andconvenient identification of cad/cad-n1; cad/cad and cad-n1/cad-n1 pinetrees. The kit may be any kit useful for detecting the presence(depicted in SEQ ID NO:3) or the absence (depicted in SEQ ID NO:1) ofthe two base pair adenosine insertion in the fifth exon of the CAD gene.The kit may be a primer extension kit, a PCR kit or a DNA sequencingkit. All of the kits include primers useful in the various detectionassays such as those described herein. The kits would also includebuffers, nucleotides and directions for use. The invention will bebetter understood be reference to the following non-limiting Example.

EXAMPLE

Materials and Methods

Plant Material

Four plant material sources were used for the identification and testingfor the presence of the cad-n1 allele: (1) A panel of 32 loblolly pinemegagametophytes (Weyerhaeuser Company Federal Way, WA, USA), includingone megagametophyte from clone 7-56, was used for SNP discovery withinthe cad gene, (2) 167 clones (CellFor Inc., Vancouver, BC, Canada)resulting from nine crosses, using clone 7-56 or 7-56 offspring asparents, was used for testing the FP-TDI assay, (3) A selection of 242first-generation clones (North Carolina State University CooperativeTree Improvement Program and Weyerhaeuser Company Federal Way; Wash.,USA) from the natural range of loblolly pine was used for estimating thefrequency of the cad-n1 allele, and (4) 96 progeny from the VERIFICATIONpopulation (Brown et al., submitted) of the QTL pedigree (Groover etal., 1994) was used for investigating the cad-psl locus.

Seeds from loblolly pine clone 7-56 were germinated and the haploidmegagametophytes were removed for CAD isozyme analysis or DNAextraction. CAD isozyme assays were performed as described by MacKay etal. 1995. All DNA extractions were performed using the Plant DNAeasy kit(Qiagen, Valencia, Calif., USA) in either the single tube or 96-wellformat.

All primers for PCR and their purpose are described in Table 1 and theirrelative position within the cad gene shown in FIG. 1.

TABLE 1 Sequence of oligonucleotide primers listed by their function.Purpose Forward primer Reverse primer Discovery CADF2- CADR2- (PCR andCCTCTGTTATGTGCAGGGGTTACA CGAAGTGCAACGGCTCTGG sequencing) (SEQ ID NO:9)(SEQ ID NO:10) FP- CADF8- CADR2- TDI (PCR) TGAAAAGATGATGTGCGCCAACGAAGTGCAACGGCTCTGG (SEQ ID NO:11) (SEQ ID NO:12) FP- CAD1528F-CAD1528R- TDI assay ATCCGTTGTGTTGCAGGAA GTAATCTAGGCTCTCTGCTGCTT (SEQ IDNO:13) (SEQ ID NO:14)

All PCR reactions were performed on ˜20 ng template in a total volume of25 μl. Each reaction comprised of 0.8 μM of each primer; 0.65 units ofHotStarTaq DNA polymerase (Qiagen, Valencia, Calif., USA); 1×PCR buffercontaining 1.5 mM Mg; 100 μM each of dATP, dCTP, dGTP, dTTP (AppliedBiosystems, Foster City, Calif., USA). Amplification was performed on aPTC100 thermocycler (MJ Research, Waltham, Mass., USA) with thefollowing parameters: Initial denaturation step of 95° C. for 15 min(for activation of HotStarTaq) followed by 37 amplification cycles of 30sec at 95° C., 30 sec at 60° C. and 2 min at 72° C.

DNA Sequencing and Analysis

To provide template for sequencing, 5 μl of PCR product was treated withIU of exonuclease I (USB, Cleveland, Ohio, USA) and IU of shrimpalkaline phosphatase (USB, Cleveland, Ohio, USA) and incubated at 37° C.for 1 hr followed by a heat inactivation step of 85° for 15 minutes. Theprimers that were used during PCR were also used for sequencing (SEQ IDNO:9 and SEQ ID NO:10). Cycle sequencing was performed using ABI Prismbig dye terminator mix (Applied Biosystems, Foster City, Calif., USA)using standard conditions as supplied by the manufacturer. Reactionswere run on an ABI 377 Automated DNA sequencer using standard ABIprotocols. Sequencher (GeneCodes, Ann Arbor, Mich., USA) was used toassemble sequences into a contig where polymorphic differences could beeasily visualized. The cad cDNA and translated protein sequence used foralignment in this study had the genbank accession numbers Z37992 andCAA86073 respectively. The intron and exon structure of the cad gene wasinferred from a Pinus radiata genomic sequence (AF060491).

Detection of the cad-n1 allele using Template-directed Dye-terminatorIncorporation and Fluorescence Polarization detection (FP-TDI).

Template for the assays was amplified using the primers CADF8 and CADR2(SEQ ID NO:11 and SEQ ID NO:12) as described in Template-directedDye-terminator Incorporation and Fluorescence Polarization detection(FP-TDI) the PCR section. The assay design for the forward and reversereaction is shown in FIG. 2 and the primer sequences listed in Table 1.FP-TDI reactions were performed using the Acycloprime-FP SNP detectionkit (Perkin Elmer Life Sciences, Boston, Mass.) as described by themanufacturer, except thermocycling conditions were altered to 25 cyclesconsisting of 95° C. for 15 seconds and 54° C. for 30 seconds.Fluorescence polarization was measured on a Wallac Victor² plate reader(Perkin Elmer Life Sciences, Boston, Mass.) with the manufacturer'srecommended filter sets and G-Factor calibration.

RESULTS AND DISCUSSION

Discovery of the cad-n1 Sequence Mutation

SNP discovery within the cad gene was performed on haploidmegagametophyte DNA from clone 7-56 and 31 other unrelated individuals.A two-base pair adenosine insertion was identified unique to clone 7-56,known to be deficient in CAD activity. The insertion was located in thesecond codon of exon five and creates a frame-shift that generates apremature stop codon (FIG. 1). Seventeen haploid megagametophytes fromthe heterozygous 7-56 clone were assayed by isozyme gel electrophoresisand DNA sequence analysis to confirm the sequence mutation discoveredwas associated with CAD-deficiency. In every case, the two-base pairadenosine insertion corresponded with the absence of CAD activity (datanot shown).

Genotyping of the cad-n1 Mutation by FP-TDI

Design of the forward and reverse FP-TDI assays are shown in FIG. 2.Trial testing of the assay was performed on 167 plants obtained fromnine different crosses involving clone 7-56 or progeny from 7-56.Results from a subset of 96 plants using the forward and reverse FP-TDIassay are shown in FIG. 3. Controls were included that consisted of allthree possible genotype classes and blanks that contained no DNA.Samples that did not fall clearly into a genotype cluster (1-2%) werenot scored. When both the forward and reverse reaction results werecombined, all plants were accurately assigned to a genotype class and nocontradictory genotypes were observed. The absence of homozygous cad-n1clones was expected based on the parental genotypes used to constructthe nine crosses tested.

Analyzing an indel mutation by single-base extension has the potentialfor giving a false result if a substitution occurs in the positionexamined (FIG. 2). For example, if the first nucleotide of codon 241 (G)is substituted to an adenosine (forward assay) or the first base ofcodon 240 (G) is substituted to an adenosine (reverse assay) a falsepositive result for the cad-n1 allele would occur. Both of thesepositions require nonsynonymous amino acid changes to occur, alanine tothreonine in the forward and glutamine to lysine in the reverse. Thesenonsynonymous changes were not observed in any of the clones present onthe SNP discovery panel or in a selection of 242 first-generationclones. If both the forward and reverse assay are performed, theprobability of an error occurring due to nucleotide substitutions wouldbe extremely low.

Since the FP-TDI assay is based on single-base extension it should beamenable to other platforms such as the SureScore SNP Genotyping Kit(Invitrogen, Carlsbad, Calif., USA) and SNaPSHOT (Applied Biosystems,Foster City, Calif., USA).

SureScore, an integrated system that requires no specializedinstrumentation, makes accessible genomic analysis tools that havetraditionally been out of reach for many laboratories. The SureScore Kitincludes primer design software, a 96-well assay kit, and data analysissoftware. The primer design software is used to design amplication andSNP-IT capture primers. The kit allows for genotyping to be conducted onup to 96 samples per SureScore strip-well plate, and commonly availableequipment such as a 96-well plate washer and reader can be accommodated.Once the assay is completed, the kit provides data analysis software tointerpret experimental results

The single base extension reaction for the FP-TDI assay utilizes aninternal extension primer, which is designed so that its 3′ end annealsadjacent to the polymorphic base-pair. The reaction is essentially asequencing reaction containing only dye-terminator nucleotides. Sincethere are no typical nucleotides, all that can occur is the addition ofa single fluorescently-labeled dideoxynucleotide (F-ddNTP), which thencannot be extended further. In the FP-TDI assay, the identity of thebase added (or bases if a heterozygote) will be discerned via measuringfluorescence polarization.

Primers and dNTPs left over from the original PCR are removed ordegraded before running the singe-base extension reaction. Residual PCRprimers are problematic because they can compete with the extensionprimer, effectively extending multiple targets, which would ruin theresults. Residual dNTPs are problematic because they can allow extensionto proceed beyond a single base.

The SNaPSHOT® system works by single base extension and then gelelectrophoresis on a sequencer such as those provided by ABI.

Frequency of the cad-n1 Allele

Frequency of the cad-n1 allele was estimated by analyzing the 242 firstgeneration clones that were distributed across the present-day range ofloblolly pine (from Texas to Florida and extending north to Delaware).The mutation was not detected in any of the clones analyzed using theforward FP-TDI assay, confirming the rareness of this mutation. Thefrequency of cad-n1 might be higher in some populations, such as in theregion where 7-56 was discovered (Williamsburg, N.C., USA), however muchmore extensive sampling would be required.

The frequency of cad-n1 in loblolly pine breeding populations andplantations will likely increase due to the inclusion of 7-56 as anelite parent in numerous co-operative and private breeding programmes.The diagnostic tool presented here will allow breeders to rapidly screenfor the presence of the cad-n1 allele in their germplasm. Screening ofadditional loblolly pine populations could be performed to identify newselect trees harboring the cad-n1 allele.

REFERENCES

The following references cited herein are hereby incorporated byreference in their entirety.

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Dimmel, D. R., MacKay, J. J., Courchene, C., Kadla, J., Scott, J. T.,O'Malley, D. M., and McKeand, S. E. (2002) Pulping and bleaching ofpartially CAD-deficient wood. J. Wood Chem. Technol. 22, 235-248.

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Lapierre, C., Pollet, B., MacKay, J. J., and Sederoff, R. R. (2000)Lignin structure in a mutant pine deficient in cinnamyl alcoholdeydrogenase. J. Agric. Food Chem. 48, 2326-2331

MacKay, J. J., Liu, W., Whetten, R., Sederoff, R. R., and O'Malley, D.M. (1995) Genetic analysis of cinnamyl alcohol dehydrogenase in loblollypine: single gene inheritance, molecular characterization and evolution.Mol. Gen. Genet. 247, 537-545

MacKay, J., O'Malley, D. M., Presnell, T., Booker, F. L., Campbell, M.M., Whetten, R. W., and Sederoff, R. R. (1997) Inheritance, geneexpression, and lignin characterisation in a mutant pine deficient incinnamyl alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 94, 8255-8260

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MacKay, J., O'Malley, D. M., Presnell, T., Booker, F. L., Campbell, M.M., Whetten, R. W., and Sederoff, R. R. (1997) Inheritance, geneexpression, and lignin characterisation in a mutant pine deficient incinnamyl alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 94, 8255-8260

Pilate, G., Guiney, E., Holt, K., Petit-Conil, M., Lapierre, C., Leple,J., Pollet, B., Mila, I., Webster, E. A., Marstorp, H. G., Hopkins, D.W., Jouanin, L., Boerjan, W., Schuch, W., Comu, D., and Halpin, C.(2002) Field and pulping performances of transgenic trees with alteredlignification. Nat. Biotechnol. 20, 607-612.

Ralph, J., MacKay, J. J., Hatfield, R. D., O'Malley, D. M., Whetten, R.W., and Sederoff, R. R. (1997) Abnormal lignin in a loblolly pinemutant. Science 277, 235-239

Wu, R. L., Remington, D. L., MacKay, J. J., McKeand, S. E., andO'Malley, D. M. (1999) Average effect of a mutation in ligninbiosynthesis in loblolly pine. Theor. Appl. Genet. 99, 705-710.

1. An isolated oligonucleotide selected from the group consisting of SEQID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
 2. Akit for the detection of the null CAD allele (cad-n1) in loblolly pinecomprising the isolated oligonucleotide of claim
 1. 3. The kit of claim2 further including materials to perform PCR reactions.
 4. The kit ofclaim 3 wherein said materials to perform PCR reactions include aplurality of PCR primers wherein said plurality of PCR primers are SEQID NO:11 and SEQ ID NO:
 12. 5. The kit of claim 2 further including oneor more buffers.
 6. The kit of claim 2 further including directions forusing the kit.
 7. The oligonucleotide of claim 1 wherein theoligonucleotide is SEQ ID NO:9.
 8. The oligonucleotide of claim 1wherein the oligonucleotide is SEQ ID NO:11.
 9. The oligonucleotide ofclaim 1 wherein the oligonucleotide is SEQ ID NO:12.
 10. Theoligonucleotide of claim 1 wherein the oligonucleotide is SEQ ID NO:13.11. The oligonucleotide of claim 1 wherein the oligonucleotide is SEQ IDNO:14.